First report of Przhevalskiana silenus derived recombinant hypodermin C based indirect ELISA for serodiagnosis of goat warble fly myiasis

Goat warble fly infestation (GWFI) is a subcutaneous myiasis caused by larvae of Przhevalskiana silenus, an insect belonging to the order Diptera. The diagnosis of GWFI is challenging in the early larval instars (L1 and L2) as they are occult under the skin and hair coat causing prolonged economic loss in form of meat and hide damage. This necessitates early diagnosis for disease control at herd level and its prophylactic management to prevent economic losses. Hypodermins, a class of serine proteases from Hypoderminae subfamily have been used as serodiagnostic antigens for the past four decades for diagnosis of warble fly myiasis. In this study,the immunodominant antigen Hypodermin C (HyC) from P. silenus has been recombinantly expressed in E. coli and immunogenic characterisation of expressed protein was done. The protein shows hallmark residues in conserved cysteine and catalytic triad typical of serine proteases along with similar profile of immunoreactivity towards Hypoderminae infestation. The present study reports an optimised indirect-ELISA based on recombinant HyC derived from P. silenus for early diagnosis of GWFI. The optimised indirect ELISA provides a sensitive and specific immunodiagnostic for mass surveillance of the GWFI with diagnostic specificity and sensitivity of 96% and 100%, respectively and not showing any cross reactivity against other important parasitic and bacterial diseases of goats. This study presents the first report of indirect ELISA based on recombinant Hypodermin C antigen derived from P. silenus for the serosurveillance of goat warble fly disease.


Materials and methods
Parasite, cells and serum samples. First stage larvae were collected from the subcutaneous tissues of infected goats at the municipal abattoir of Jammu (India), washed with PBS, and identified as per keys of Zumpt 1 and stored at − 80 °C for RNA isolation. E. coli BL21 (DE3)pLysS cells, pET32a(+) vector(Novagen) were used in expression study. Physicoclinical observation was kept as basis for determining reference serum. For sera, blood samples were collected from field and slaughterhouse and marked as positive and negative based on larval detection by palpation in live animals and by carcass examination.Serum samples from animals testing positive for coenurosis, paratuberculosis, enterotoxaemia, oestrosis, coccidiosis, cryptosporidiosis, haemonchosis, monieziosis, fasciolosis, amphistomosis and brucellosis were used in this study.

Construction of expression cassette in pET32a(+) vector.
Total RNA was isolated from L1 stage larvae of P. silenus using RNeasy mini kit (Qiagen, Germany) as per manufacturer protocol. The cDNA was synthesized from total RNA using oligodT primer following the protocol described in cDNA synthesis kit (Revert Aid First strand cDNA synthesis kit, Thermo Scientific). The HyC gene of P. silenus was amplified using HyC gene of cattle warble fly (H. lineatum) specific expression primers based on the published sequences of HyC (EMBL Accession Number X74306) (AAGGA TCC ATA ATC AAT GGA TAC GAA G and ATCTC GAG TTA AAA TAT TAT ACC AGT ATT TTG ). The gene was amplified using a 25 µL reaction mixture containing 2 µL of cDNA, 2.5 µL of 10X Ex Taq buffer (TaKaRa,USA), 1 µL each of forward and reverse primers (10 pmol/µL), 1 µL of 10 mM dNTP mix, 0.5 µL of Ex Taq DNA Polymerase (TaKaRa, USA), and 17 µL nuclease free water. PCR reaction was carried out at initial denaturation of 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 72 °C for 1 min and a final extension at 72 °C for 10 min.
The amplified PCR product was analysed by 1% agarose gel electrophoresis. The amplified product was purified using Wizard™ SV Gel and PCR Clean-Up System (Promega, USA), directionally cloned into pET32a(+) expression vector at BamHI and XhoI restriction enzyme sites and transformed into TOP10 E. coli cells. The recombinant clone was selected on LB agar plate containing Ampicillin (100 μg/mL). The recombinant plasmid containing gene of interest was confirmed by colony PCR using gene-specific primers and restriction enzyme digestion. The recombinant plasmid was isolated using Plasmid extraction kit and sent for nucleotide sequencing to Agrigenome Pvt. Ltd (Kerala). The sequence received was primarily analysed using BioEdit software and submitted to Gen Bank. Further, the protein encoding nucleotide sequence was translated in silico using the Edit

Expression and purification of recombinant protein. The recombinant HyC plasmid was trans-
formed into E. coli BL21(DE3)pLysS host cells and further confirmed by colony PCR. The positive clone was grown in LB broth containing ampicillin and chloramphenicol at 37 °C for overnight. The fresh LB broth was inoculated at 1:100 with overnight culture and incubated at 37 °C at 200 rpm till the culture reached an OD 600 0.4-0.6. Henceforth, the expression was induced with 1 mM IPTG and harvested 1 mL culture at hourly interval up to 16 h. The collected fraction was pelleted by centrifugation and level of expression was analysed by SDS-PAGE. The rHyC protein was produced in bulk and the pellet was resuspended into denaturing buffer (8 M Urea) and sonicated at 25% amplitude with pulse of 15 s for 5 min. The lysate was clarified and the supernatant was purified by using Ni-NTA affinity chromatography under denaturing condition (8 M Urea). The Ni-NTA superflow gravity column was equilibriated with denaturing binding buffer containing 8 M urea in phosphate buffer (pH 7.8) followed by binding of clarified lysate with Ni-NTA Superflow® resin (Qiagen). The proteinbound resin was washed in subsequent steps with wash buffer at pH 6.0 and pH 5. Optimization of rHyC based iELISA. An indirect-ELISA using rHyC has been optimised and evaluated with known status of positive and negative GWFI sera to detect the anti-HyC antibodies. The optimum concentration of coating antigen, dilution of serum and conjugate, standard checkerboard titration was performed. A total of three different concentration of antigen i.e. 0.5 μg/mL, 0.25 μg/mL and 0.125 μg/mL (100 μL/well), three different dilutions of sera at 1:400, 1:800 and 1:1600 and two dilutions (1:5000 and 1:10,000) of anti-goat IgG HRP conjugate were used in checkerboard titration analysis using 5% SMP as blocking buffer. The maximum differentiation between positive and negative sera (P/N) measured at A 490 was selected for further analysis.
In brief, 96-well microtiter plate (Costar 3590, Corning-USA) were coated with 100 μL of rHyC (0.5 μg/ mL) in 0.05 M carbonate bicarbonate buffer (pH 9.6). The plate was incubated at 4 °C overnight. The wells were washed twice with PBS-Tween 20 (0.075%), and then once with PBS, blocked with 5% skimmed milk prepared in PBS (pH 7.4) and incubated at 37 °C for 1 h. After washing, 100 μL of known positive and negative serum sample diluted in PBS (1:400) were added and incubated at 37 °C for 1 h. After washing the plate three times, 100 μL of anti-goat IgG HRP-conjugate (Sigma Aldrich, USA) at 1:10,000 dilution in PBS (pH 7.4) was added to each well and incubated at 37 °C for 1 h. Following washing, 100 μL of O-phenylenediamine dihydrochloride (OPD) substrate (Sigma Aldrich, USA) dissolved in citrate-phosphate buffer (pH 5.0) with 30% H 2 O 2 was added to individual wells. The reaction was allowed to develop for 10 min in dark and reaction was stopped by adding 50 μL of 1 M H 2 SO 4 to each well. Absorbance at 490 nm was measured by using microplate reader (iMarkmicroplate reader, Biorad).

Evaluation of rHyC based iELISA.
After optimisation of the iELISA, serum samples with known status (15 positive and 25 negative) were screened in duplicate for determining the cut-off value. The physicoclinical evaluation was taken as a reference to distinguish the positive and negative samples. Negative reference serum samples were received from animals that had never grazed and belongs to Punjab state of North India, where GWFI has never been reported. The cut-off value was evaluated by the Receiver Operating Characteristic (ROC) curve, using MedCalc software, Mariakerke, Belgium 40 . Based on the checkerboard titration results, ROC curves and areas under the curve were plotted, and the sensitivity and specificity of iELISA were calculated using interactive dot diagrams. Cut-off value was calculated according to the Youden index (Youden = Se + Sp − 1) and used for immunodiagnosis of goat warble fly. In order to rule out the cross reactivity of optimised rHyC based iELISA with other economically important parasitic and bacterial diseases of goats, known sera positive for oestrosis, monieziosis, coccidiosis, fasciolosis, haemonchosis, amphistomiosis, coenurosis, cryptosporidiosis, Johne's disease, brucellosis and enterotoxaemia were also screened.

Results
Cloning and bioinformatics analysis of rHyc from P. silenus. PCR amplification of the HyC from P.
silenus was obtained by using cDNA template produced from L1 larval instars with oligodT primers. The amplified product of expected size 706 bp was confirmed on 1% agarose gel electrophoresis (Fig. 1). The gel purified PCR product was cloned into pET32a( +) vector at BamHI and XhoI restriction enzyme sites and transformed into TOP10 E. coli cells. The transformants were screened on LB agar plates containing ampicillin. The recombinant plasmid was characterised by colony PCR and RE digestion that confirmed the presence of target gene and its orientation into the vector. The recombinant plasmid was isolated, sequenced and the gene sequence was submitted to GenBank (Accession number MZ407908). Sequence similarity searches in Blastn revealed that the HyC from P. silenus (HyC_PS) was 86% identical to HyC of H. lineatum and H. bovis. The alignment of HyC_PS with HyC protein reported from H. lineatum (HyC_HL), H. bovis (HyC_HB) and H. diana (HyC_HD) revealed 18.695% variation in the polypeptides, with 43 substitutions out of total 230 amino acids (X74306.1) (Fig. 2). The HyC_PS with 232 residues with a predicted molecular weight of 25.150 kDa contains 22 strongly acidic, 12 strong basic, 79 hydrophobic residues and 78 polar amino acid residues with a predicted pI of 4.569. The molecule contains all the six cysteine residues as shown in sequence logo, at conserved position resulting in three disulphide bonds. The conserved residues of catalytic triad as His-45, Asp-88, Ser-180 are also present in the protein representing the functional feature of serine proteases (Fig. 2).
Secondary structure prediction analysis indicated the trypsin domain structure for HyC with presence of three chymotrypsin peptidase regions in the HyC amino acid sequence. Secondary structure analysis also revealed that HyC_PS consists of 9% alpha-helix, 43% beta-strand and 16% unstructured coil regions, respectively 41 .

Expression of rHyC and its characterisation. The verified recombinant clone with HyC gene in BL21
cells was used for expression study. The expression of protein was induced through 1 mM IPTG at 37 °C and the samples collected at hourly interval were analysed by SDS-PAGE to check the maximum level of expression for the construct. The different fractions were compared for expression with the uninduced sample showed about 45 kDa of protein overexpressed upon 6 h post induction, maximally. The protein separated in SDS-PAGE was transferred on nitrocellulose membrane (NCM) for blotting and expressed recombinant HyC protein was confirmed by Ni-NTA HRP conjugate.
Purification and immunoreactivity of rHyC. After optimisation, expression was scaled up by increasing the culture volume and purified under denaturing condition using Ni-NTA affinity chromatography. The eluted fractions were analysed by SDS-PAGE that indicated the presence of single band with about 45 kDa size with near homogeneity (Fig. 3). The yield of expressed fusion protein was at 2.8 mg/L of induced culture. The antigenicity of purified recombinant protein was confirmed by immunoblotting using GWFI positive, negative serum and Oestrosis positive sera. Recombinant HyC specificity was confirmed to GWFI serum. A distinct band www.nature.com/scientificreports/ of 45 kDa was observed with the expressed protein reacting strongly and specifically with the GWFI positive serum. No reactivity was observed with goat negative and oestrosis positive sera (Fig. 4).
Optimisation and evaluation of rHyC iELISA. Checkerboard titration revealed that at antigen concentration of 0.5 μg/mL (100 μL/well), serum dilution of 1:400, conjugate dilution of 1:10,000 with 5% SMP as blocking buffer were found to give maximum difference in the reactivity between positive and negative serum. ROC analyses using sera from 15 infested and 25 non-infested goats revealed an area under curve (AUC) of 0.992 (P < 0.001) for rHyC iELISA. Relative diagnostic sensitivity and specificity calculated by interactive dot analysis of MedCalc software were 100% and 96%, respectively (Fig. 5). A cut off value (OD) of 0.4835 for rHyC iELISA was fixed from the ROC curve and interactive dot diagram (Fig. 6) and used for serological screening of the random samples. The expressed protein showed strong reactivity with GWFI positive serum whereas it did not show any significant reactivity with other economically important parasitic and bacterial diseases of goat (Fig. 7). Further, screening of random serum samples collected from different parts of Union Territory of Jammu and Kashmir, North India were tested with optimised ELISA and the corrected OD values (Mean OD of duplicates − Mean     www.nature.com/scientificreports/ OD of antigen blank) were calculated to differentiate between infested and healthy animals. Out of 421 serum samples screened through optimised iELISA for detecting anti-HyC antibodies, 59 were found to be positive and 362 tested negative for GWFI (Fig. 8).

Discussion
Goat warble fly disease is the cause of major economic losses to goat farmers with high prevalence in Mediterranean and Indian subcontinent. GWFI has been reported with varying prevalence rates in different countries. In North India, the disease has been reported to have a prevalence rate varying between 13 and 56.5% in Jammu region whereas the prevalence varies in Pakistan (5-75%), Turkey (53-94%), southern Italy (20-90%), Iran    30,37,43 . Hypodermin A, B and C are serine proteases from Hypoderminae insects which are the major immunogens in case of larval infestations. Hypodermins are reported to show overexpression in L1 larval instar as compared to L2 and L3 stages 32 . Hypodermin C has been established as the major antigen for serological detection of hypoderminae myiasis and shows cross-reactivity among Hypoderminae members (H. bovis, H. lineatum, H. tarandi, H.actaeon, H. sinense and P. silenus) due to shared epitopes as evident by several studies of cross reactivity and ELISA 19,21,35,38,44,45 . The HyC derived from P. silenus has shown several substitutions interspersed in the whole sequence. On comparison with other serine proteases reported from H. lineatum, H. bovis and H. diana, the HyC presents all the conserved features attributed to serine protease activity such as conserved cysteine residues and the catalytic triad at the designated sites (His-45, Asp-88, Ser-180) 15,28,32 . Although, the HyC protein derived from P. silenus show considerable variations in residues (18.695%, 43 substitutions/230 aa), the three dimensional structure shows strong homology in the predicted structure as compared to that of HyC from H. lineatum 16 .
ELISA has been promoted as surveillance tool in diagnosis of warble fly in various countries 19,21,[46][47][48][49] . It is preferred over palpation or examination of carcass at slaughter 49 . Serology provides more sensitive detection of exposure to warble fly myiasis as a number of animals might resolve larvae over a period and can only present serological detection 49 .Thus, ELISA provides a tool of warble fly infestation study in occult stages as well as in case of early resolution of the larvae before warble development on the dorsum in case of GWFI. The antibody kinetics against H. lineatum, H. bovis, P. silenus, and H. tarandi in their hosts have revealed that it takes about one month to reach antibody peaks in the host sera, which implicates the utility of ELISA as diagnostic tool at L1 stage of infestation 20,29,30,37 .
Several in-house ELISA assays are used in different countries which are primarily based on native antigen derived from H. lineatum and H. bovis larvae. Native antigen and purified antigen preparations provide challenge in standardisation due to purification and requirement of larvae repeatedly. Recombinant protein may serve as antigen for better reproducibility and standardisation for a diagnostic test. Also, since the parasite availability for native antigen is decreasing and fluctuating upon progressive control measures in many countries, recombinant proteins serve as constant source of antigen for diagnostic evaluation of the population 33 . Recent study has provided rHyC based indirect ELISA derived from H. bovis for the diagnosis of hypodermosis in cattle 28 whereas another study developed in-house ELISA protocol for the detection of GWFI 9 which are based on the use of a larval crude lysate as antigen. A summary of selected ELISA for Hypoderminae myiasis detection is provided in Table 1. In natural infestations, larval development is not synchronous as individual hosts may have multiple oviposition events and this apparently causes episodic release of HyC limiting the utility of antigen-capture ELISA 31,50,51 . On the other hand, in case of competitive ELISA, sample dilution is permitting lower coverage of the herd surveillance in case of pooled sampling 36 .
The present study provides first molecular characterisation of HyC protein, the major antigen for serodiagnosis of warble fly myiasis, in case of goats, caused by P. silenus. The study characterised the HyC of P. silenus by cloning and heterologous expression in E. coli and assessed its immunoreactivity with GWFI positive sera along with other economically important goat diseases.
The HyC derived from P. silenus was amplified with gene specific primers and cloned into pET 32a(+) vector. The expected size of target protein is about 25 kDa whereas the complete construct used in this study presents around 45 kDa product as a fusion protein with thioredoxin tag and the same size was obtained as expected in SDS-PAGE analysis upon induction. The optimum level of expression was achieved at 6 h post-induction after which there was no improvement in the expression level. Purification was done under denaturing conditions with 8 M Urea in PBS with elution based on pH gradient in acidic condition. At a yield of 2.8 mg/L of induced culture, the recombinant fusion protein may be sufficient to screen approx. 2,800 serum samples in duplicate with the optimised protocol. The purified HyC protein reacted strongly with GWFI positive serum in ELISA format www.nature.com/scientificreports/ at antigen concentration of 0.5 μg/mL (100 μL/well) at 1:400 dilution of serum identified by checkerboard titration. The present ELISA showed no cross reaction with any other major parasitic and bacterial diseases of goats. The rHyC of P. silenus as an antigen in the ELISA provided a diagnostic sensitivity and specificity of 100% and 96% respectively, for serodiagnosis of goat warble fly disease The other iELISA established with rHyC derived from H. lineatum exhibited sensitivity of 85% and specificity of 98.2% whereas iELISA based on rHyC from H. bovis has shown sensitivity of 90% and specificity of 100% 25,27 . Also, the study comparing utility of native HyC (nHyC) and rHyC as antigen in ELISA for detection of anti-Hypoderma antibodies has shown a sensitivity of 95.8% and specificity of 95.7% for rHyC and 98.2% sensitivity as well as specificity for nHyC based iELISA 32 . On the other hand, competitive ELISA for detection of hypodermosis showed a sensitivity and specificity of 92.5% and 98.5%, respectively 36 . The iELISA based on rHyC is optimised with a high sample dilution rate at 1:400 for GWFI serum samples. This provides an opportunity for a standardised test as a tool for mass surveillance of GWFI at national eradication program. Also, the high standard dilution rate permits scope of using pooled serum samples for the assessment of herd screening for GWFI 52,53 . It has also been used for the screening of random serum samples from different parts of Union Territory of Jammu and Kashmir, North India. The optimised assay is highly specific and sensitive for detection of antibodies against goat warble fly disease.
ELISA test has been adopted for mass epidemiology of hypodermosis as a tool of monitoring at national level eradication programs in countries like France, the UK, Belgium and Germany 37 . ELISA has proven an effective tool to establish antibody kinetics in both bovine hypodermosis and GWFI. This has further assisted the monitoring initiatives to facilitate appropriate timing of sample collection according to particular geographical and agroclimatic zones 8,9,19,54 . Durable control of GWFI requires repetitive monitoring for the detection of any reappearance of the disease in any geographical region. Serological monitoring is vital tool for aid in policy implementation of control and eradication programmes through the use of systemic insecticides 52,53 . As it is evident by immunological studies that Hypoderminae species do not provide complete protective immunity, it is plausible that constant monitoring and surveillance along with prophylactic treatment remains the practical solution for durable control of goat warble fly as has been observed in cattle hypodermosis 49,51 .

Data availability
All data generated or analysed during this study are included in this published article (and its supplementary information files). The sequences used for comparison are available under the accession numbers: X74306; MK473847; EU999953; MZ407908 from NCBI database at https:// www. ncbi. nlm. nih. gov/. www.nature.com/scientificreports/